Urist published first studies on bone formation by autoinduction and disclosed new protein factors involved in therein in 1965 [Urist, M. R., Science 150 (1965) 447-454]. The bone inducing principle could not be attributed to a single protein, but to a group of proteins, where each group member alone is sufficient to induce heterotopic ossification (HO). This started a long search for the bone inducing principle, and it took over twenty years before the first cDNAs encoding bone morphogenetic proteins or BMPs were cloned [Wozney, J. M., et al., Science 242 (1988) 1528-1534]. Based on sequence and structural analysis, additional proteins like the growth and differentiation factors (GDFs) could be linked to the original BMPs. Together they build the BMP-family with more than thirty members [Reddi, H., Cytokine & Growth Factor Reviews 8 (1997) 11-20], which belongs to the TGF-β-super-family.
The BMP-family is divided to subfamilies including the BMPs, such as BMP-2 and BMP4, osteogenic proteins (OPs), such as OP-1 or BMP-7, OP-2 or BMP-8, BMP-5, BMP-6 or Vgr-1, cartilage-derived morphogenetic proteins (CDMPs), such as CDMP-1 or BMP-14 or GDF-5, growth/differentiation factors (GDFs), such as GDF-1, GDF-3, GDF-8, GDF-9, GDF-11 or BMP-11, GDF-12 and GDF-14, and other subfamilies, such as BMP-3 or osteogenin, BMP-9 or GDF-2, and BMP10 (Reddi et al., supra).
One common feature shared by the BMP-family members and other members of the TGF-β super-family is the overall folding topology which resembles a hand exhibiting the victory sign, with the wrist representing the central alpha-helix, two fingers representing two anti-parallel β-sheets, and the palm representing the cysteine-knot region [Scheufeler, C. et al., J. Mol. Biol. 287 (1999) 103-115]. The cysteine-knot consists of three intrachain disulfide bridges and is the main stabilizer of the 3-D structure, and it is so effective that biological activity is preserved even after extensive exposure to low pH, urea or guanidine hydrochloride treatments [Sampath, T. K., and Reddi, A. H., Proc. Natl. Acad. Sci. USA 78 (1981) 7599-7603]. Further stabilization of the topology is achieved by the dimerization of two monomers, creating an internal hydrophobic core (Scheufeler, C. et al., supra). The overall dimeric structure is also necessary for the biological action, such as osteoinduction, as it enables the simultaneous binding of a dimeric molecule to the corresponding receptor, for instance the simultaneous binding of a BMP-dimer to a type I and a type II serine/threonine receptor forming a heterotetramer, triggering a signal cascade via the phosphorylation of Smads [Yamashita, H. and Miyazono, K., Nippon Rinsho 57 (1999) 220-226].
Results based on the protein and nucleotide sequences of the BMPs have revealed that the morphogenesis of bone, comprising chemotaxis, mitosis, and differentiation, is governed by the action of the BMPs. It has also been shown that the effects of the BMPs are not limited to bone and cartilage. In the early stage of embryogenesis the BMPs rule the formation of the entire body plan and specify the tissue types and axes. In an adult, the BMPs affect to the ability of bones to repair successfully [for review, see Wozney, J. and Rosen, V., Clin. Orthop. Rel. Res. 346 (1998) 26-37]. This aspect of the BMP action together with its osteoinductive power has led to the idea to use the BMPs in the treatment of patients for the enhancement of fracture healing and the augmentation of bone. Initial studies performed with native BMP preparations from human bones [Johnson, E. E. et al., Clin. Orthop. 250 (1990) 234-240; Clin. Orthop. 277 (1992) 229-237] and bovine bones [Sailer, H. F. and Kolb, E., in Bone Morphogenetic Proteins: Biology, Biochemistry and Reconstructive Surgery, pages 207-230, Lindholm, T. S., ed., R. G. Landes Co, 909 Pine St, Georgetown, Tex. 78626, 1996] revealed the efficacy of the BMPs in orthopaedic and cranio-maxillofacial surgery, especially in difficult situations.
Another aspect of the BMPs is their interaction in the manifestation of heterotrophic ossification (HO), which has created expectations to develop inhibitors of the BMPs and use them as therapeutic agents in HO. HO is a frequent complication in patients who have suffered head and neck traumas, traumatic acetabular fracture, or undergone total hip replacement. It is a process of bone formation at ectopic sites, such as muscle and connective tissue, that can lead to decreased mobility, pain, or even total ankylosis, predominantly in hip and elbow joints [for review see: Nilsson, O. S., Acta Orthop. Scand 69 (1998) 667-674]. As the elderly population increases and the number of the total hip arthoplastic operations rises, the number of patients suffering from HO can be expected to grow. Additionally, HO is manifested in some inherited diseases, such as fibrodysplasia, or acquired bone forming lesions, such as spinal hyperostosis, myelopathy and spondylitis ankylosans, in which no curable treatment is available or a surgical operation is the only means of treatment.
Unfortunately, up to now, the discovery of the BMPs has had no impact on the medical treatment of patients, despite encouraging initial results. This is mainly because sufficient amounts of the BMPs or their inhibitors are not available. The purification of the BMPs from natural sources does not result in sufficient amounts of the proteins for treatment purposes, nor are the yields of recombinant human BMPs (rhBMPs) produced in mammalian expression systems satisfactory. Furthermore, individual rhBMPs are not as effective as native human BMP preparations: it has been shown that the latter are ten times more effective in the induction of ectopic bone formation than pure human recombinant BMPs [Bessho, K., et al., Br. J. Oral. Maxillofac. Surg. 37 (1999) 2-5]. Finally, a major problem for a routine application of the BMPs in patients is the lack of a suitable BMP delivery system. The BMPs must be administered with a carrier, since their administration for instance by an injection results in an instant onset of the BMP degradation. This is strikingly contradictory to the BMP expression profiles which show that during fracture healing for instance BMP-2 is expressed up to 14 days after the fracture occurred [Kitazawa, R. et al., Acta Histochem. Cytochem. 31 (1998) 231-236].
Obviously there exists an urgent need for new approaches in the utilization of the BMPs. The present invention provides a solution to overcome the disadvantages and drawbacks described above.
A purpose of the present invention is to provide means for the utilization of the bone forming inductive activity of the BMPs in the treatment of patients in orthopaedics, dentistry and other fields in medicine.
Another purpose of the invention is to provide means for the utilization of the bone forming inhibitory activity of the BMPs in the treatment of patients in orthopaedics and other fields in medicine.
Specifically, a purpose of the present invention is to provide a pharmaceutical composition that would be useful in the treatment of diseases in which the enhancement of fracture healing and the augmentation of bone is desired. Such a composition would improve the recovery of a patient who, e.g., has undergone a surgical bone operation or suffers from accidental bone fracture. In addition, such a composition would find use in the integration of dental implants, in the filling of tooth sockets following extraction, in the alveolar ridge augmentation, in the sinus floor elevation, and in the healing of non-unions.
Additionally, another special purpose of the present invention is to provide a pharmaceutical composition that would be useful in the treatment and prevention of heterotrophic ossification and other diseases involving undesired bone formation. Such a composition would significantly add to the options that now are available in the treatment of HO, and would lack the side effects of the non-steroidal anti-inflammatory drugs (NSAIDs) and the radiotherapy, which at present are the alternative methods of treatment.
Still another purpose of the invention is to provide new methods for the treatment of orthopaedic patients, which would lead to an accelerated and permanent recovery.
Still another purpose of the invention is to provide new methods for the treatment of dental patients, which would lead to an improved dental health.